131 results about "Cell factory" patented technology

The invention discloses a brewer's yeast engineering bacterium for producing valencene and a construction method and application of the brewer's yeast engineering bacterium. Overall inhibiting factorgenes in a yeast genome are subjected to knockout, a squalene synthetase gene is subjected to down reduction of expression, valencene synthetase is expressed, HMG-CoA reductase is overexpressed, the promoter of the valencene synthetase is optimized, and therefore the valencene is obtained. The key gene in the metabolic pathway is overexpressed through a free vector, the metabolic flux of the valencene in the synthetic route is increased, and the yeast cell factory is optimized; in combination with the suitability research of the promoter of the valencene synthetase, the brewer's yeast engineering bacterium obtained through culture optimization achieves efficient production of the valencene, and the dry cell weight yield of the valencene can reach 124.40 mg/g; the brewer's yeast engineeringbacterium has a wide industrial application prospect and positive economic and social development significance.

The invention discloses a method for applying a multi-axis robot to cell factories to prepare biological products. The problems that in the prior art, a large amount of manual operation is needed, theconstant temperature room environment is difficult to control, thus the adverse effect on growing and amplification of cultures is caused, and the production cost is high are solved. A plurality of sets of culturing frames, arm operating openings and pipeline connecting openings cooperate with the multi-axis robot to achieve operations such as grabbing, moving, overturning and waggling of specialstructure cell factory frames to replace operating processes adopting manpower and other fixed equipment, wherein the culturing frames are sequentially arranged in a fan shape in a constant temperature room, and the arm operating openings and the pipeline connecting openings are formed in an observation window between the constant temperature room and an operation room. According to the method, the cell factories are intensively cultured and operated, operation does not need manual pushing and does not need to leave the constant temperature room, thus the culturing environment is easy to control, changing of the culture temperature is small, modular efficient production is facilitated, the product consistency is good, the pollution rate is low, and the overall operating cost can be saved.

The invention belongs to the technical field of gene editing, and particularly relates to a Zymomonas mobilis genome editing method based on a CRISPR-Cas12a system and application of the method. The method for editing a Zymomonas mobilis genome is established on the basis of the CRISPR-Cas12a system with Zymomonas mobilis (ZM4) as the modulus strain, the oriented editing of the genome is realized,a gene editing tool is provided for developing the rational design of an allogenic metabolic pathway and cell factory for producing biomass fuel and biological materials in the strain, and the development in metabolic engineering and related research fields is promoted. The method is technically characterized by including the steps of establishing an inducible expression Cas12a recombinant strainand an editing plasmid containing an artificial CRISPA expression unit, designing guiding RNA, connecting the guiding RNA to the editing plasmid after annealing the primer sequence of the guiding RNA, and shifting a target plasmid into a competent cell for expression editing.

The invention relates to a method for scale-up culture of animal cells. The method includes the following steps of firstly, culturing animal cells in a cell factory; secondly, digesting the cultured animal cells, namely removing culture solution, and washing the cultured animal cells by pH7.4-8.0 washing solution; removing the washing solution by adding pH7.4-8.0 digestive solution 20-80mL per layer, allowing the digestive solution to evenly flow to each layer to digest the cells at 36-38 DEG C, removing partial digestive solution when more than 60% of cells fall off from the cell culture surface, and continuing to digest by the residual 10-35% of digestive solution; and adding fresh cell culture solution containing serum or digestive solution inhibitor to finish digesting and performing cell suspension again, and adding the culture solution for once to three times repeatedly; and thirdly, inoculating obtained cell suspension to a bioreactor for culture.

The invention provides a method of preparing a hepatitis A inactivated vaccine, which comprises the following steps of inoculating mixed and absorbed hepatitis A virus strain SH and a human embryo diploid cell MRC-5 to hepatitis A virus propagated in a cell factory, digesting the cell in the virus propagation peak to obtain cell virus liquid, removing impurity proteins of the cell by ultrasonication, chloroform extraction and ultrafiltration through ultrafiltration membranes, degerming and filtering by gel filtration chromatography and purification, and absorbing by an aluminium hydroxide adjuvant so that the hepatitis A inactivated vaccine is prepared. The result of in vivo effectiveness experiments performed on a mouse shows that the hepatitis A inactivated vaccine prepared by the method of the invention has higher ED50 and better immunogenicity than the contract strain. The method of the invention can improve the safety of the vaccine, simplify the technique, shorten the productionperiod, reduce the production cost, and realize the scale production of the hepatitis A inactivated vaccine.

The invention provides a cell-factory-based measles virus stock solution and a measles-series attenuated live vaccine preparation. A preparation method of the measles virus stock solution includes selecting SPF chick-embryo cells, and adding cell culture fluid to prepare cell suspension liquid, and adding the cell suspension liquid into a cell factory; inoculating the cell factory with working seeds of measles viruses and the cell suspension liquid according to the ratio of the working seeds to the cell suspension liquid being (0.005-0.05):1, and standing and cultivating the cell factory at the temperature of 33+/-1 DEG C for 3-4 days, pouring out archeocyte culture fluid, and replacing with fresh cell growth liquid for continuous cultivation; during cytopathy, collecting single measles virus liquid step by step. In an equal production scale, the batch number of cell dissociation is reduced, and the high-titer measles virus liquid is obtained, so that quality uniformity of measles-series vaccine products is improved effectively, and product yield is increased.

The invention relates to the field of culture containers, in particular to a novel multi-layer adherent cell culture container structure. The novel multi-layer adherent cell culture container structure comprises a multi-layer cell factory main body, a bayonet cover and a breathable cover, wherein the interior of the multi-layer cell factory main body is provided with a plurality of groups of multi-layer culture surfaces in an equally-spaced mode from top to bottom; multi-layer culture surface fixed grooves are formed in the multi-layer cell factory main body at the position close to the multi-layer culture surfaces; extension tube openings are formed in the side, close to a base surface and a back surface, of the upper end of the multi-layer cell factory main body respectively; the bayonet cover is fixedly mounted at the upper end of the extension tube opening in the side, close to the base surface, of the upper end of the multi-layer cell factory main body; the breathable cover is fixedly mounted at the upper end of the extension tube opening in the side, close to the back face, of the upper end of the multi-layer cell factory main body; the interior of the breathable cover is provided with an extension tube; and the exterior of the extension tube is provided with a breathable film. Through the application of the breathable cover, the container can be used for flooded culture, the storey height is reduced, the culture area is greatly increased under the same height, and the utilization rate of space is promoted.

The invention belongs to the technical field of biological medicament making, and discloses a method for preparing a Marek's disease liquid nitrogen vaccine (814-strain) by utilizing a cell factory and the Marek's disease liquid nitrogen vaccine (814-strain) prepared by the method. The Marek's disease liquid nitrogen vaccine (814-strain) produced by utilizing the cell factory has high quality, a high immune efficiency and a stable immune effect; in addition, the Marek's disease liquid nitrogen vaccine also has the advantages of low generation, small plaque, high plaque forming unit (PFU) content, high immunogenicity, low cost, capacity of generating immunoprotection in a relatively short time and the like.

The invention discloses a single cell factory capable of efficiently synthesizing L-phenylglycine as well as construction and application of the single cell factory and belongs to the technical fieldof microorganisms. Firstly, efficient expression of leucine dehydrogenase obtained from Bacillus cereus in escherichia coli is realized, and site-directed mutation is carried out to obtain a mutant N71S with a remarkably improved reduction property; a mutant enzyme and a formate dehydrogenase mutant are co-expressed in the escherichia coli to form an intracellular in-situ co-factor NADH (Nicotinamide Adenine Dinucleotide) circulating system; the expression amount of the formate dehydrogenase mutant is optimized and controlled through a promoter and an RBS (Ribosomal Binding Site) sequence to successfully construct a recombinant escherichia coli single cell factory; the single cell factory is subjected to whole-cell conversion to prepare the L-phenylglycine. The method disclosed by the invention has the advantages of simple and rapid conversion process, low cost, no byproduct and easiness for separation and purification; when conversion is carried out in a 5L fermentation tank for 4h, the yield of the L-phenylglycine can reach 105.7g/l, the conversion rate is 93.3 percent and the space-time yield of the L-phenylglycine is 26.3g/L; an actually practical and effective strategy is provided for industrial production of the L-phenylglycine.

Provided is a cultivation method for promoting growth of transgenic tomatoes and accumulation of astaxanthin. The cultivation method comprises the steps of taking the astaxanthin transgenic tomatoes as materials, sowing seeds of the transgenic tomatoes, transplanting seedlings growing for two months after emergency of seedlings, covering the transgenic tomato seedlings with nylon nets respectively to perform shading processing, measuring light intensity through a quantum illumination meter, performing processing respectively according to four light intensity gradients including shading 75 percent of light, shading 50 percent of light, shading 30 percent of light and performing full illumination, performing photosynthesis measurement respectively at a flowering phase and a fruit phase, harvesting the transgenic tomatoes for three times when the transgenic tomatoes are ripe, and measuring contents of the astaxanthin in the transgenic tomatoes. In the planting process, watering is performed in routine to ensure a good moisture state, cooling processing is performed through fans and wet curtains, and deinsectization and disease prevention are performed at regular intervals. According to the cultivation method, the astaxanthin transgenic tomatoes serve as cell factories to produce the astaxanthin efficiently, photosynthetic capacity of the transgenic tomatoes and astaxanthin contents in fruits are optimal under the conditions that the light intensity is 30 percent of sunlight, and the foundation is laid for establishing an artificial efficient astaxanthin-rich transgenic tomato cultivation technique system.

The invention discloses a high-flux screening method based on a biosensor, and belongs to the technical field of high-flux screening. A way of embedding biosensor-containing cells by liquid drops is adopted to carry out high-flux screening on a metabolite micromolecule 3-dehydrogenation shikimic acid cell factory to realize the efficient enrichment of 3-dehydrogenation shikimic acid high-yield bacterial strains. Compared with a single cell level screening method for combining the biosensor with a flow cytometer, on the one hand, the high-flux screening method disclosed by the invention utilizes the augmentable enrichment characteristics of cells in the liquid drops to enlarge an inter-unicellular fluorescence signal difference for N times to a difference among cell groups; on the other hand, through the characteristics that the liquid drops are used for isolating single cells and secretory products thereof, the influence of other cells and secretory products is eliminated, reductive cells and extracellular metabolite secreted by the reductive cells are truly communicated and fed back; through the above improvement, the screening sensitivity of the profitable positive clone bacterial strains can be effectively improved, and the enrichment efficiency of the profitable positive clone bacterial strains is improved.

The invention belongs to the technical field of biology, and relates to a full-automatic cell production line. The full-automatic cell production line comprises a culture zone and an operation area,; the culture zone comprises a B-level platform body, and the B-level platform body comprises a culture area, a refrigeration area and a robot provided with a motion track; the motion track of the robot is linearly arranged; the culture area, the refrigeration area and a manual delivery window are all arranged around the track and are all located in an operation area of the robot; a liquid storage table, a cell factory liquid changing device and a centrifugal bottle liquid changing device are integrated in the operation area; a transfer window is adopted between the culture area and the operation area to transfer materials; and the transfer window is provided with a transfer rotating disc and a transfer rotating disc driving mechanism, and the transfer rotating disc is provided with a zero position for the robot in the culture zone to pick and place the materials and a working position for a robotic arm in the operation area to pick and place the materials. The full-automatic cell production line is mainly used for full-automatic cell batch production.

The invention discloses a genetic engineering bacterium highly producing D-allulose and application of the genetic engineering bacterium. A food-grade strain bacillus subtilis is used as a host bacterium, D-tagatose 3-epimerase (DTEase) is efficiently expressed through gene recombination, and a cell factory for producing D-allulose by directly using D-fructose is formed. The genetic engineering bacterium highly producing the D-allulose is obtained by screening, through optimization of a fermentation process, the yield of the D-allulose reaches 4.56 g/L, and the conversion rate of a substrate is 56.26%, and the production efficiency of the D-allulose is 0.19 g/L*h.

The invention relates to a method for purifying a slow virus. The method comprises the following steps: (S10) providing a cell culture containing the slow virus; (S20) carrying out centrifugal concentration on the cell culture, and filtering to obtain virus suspension; (S30) purifying the virus suspension by virtue of anion exchange chromatography; and (S40) purifying the virus suspension by virtue of gel filtration chromatography. A process for purifying the slow virus is easy to amplify, can be used for processing large amounts of virus liquid produced by cell factories or bioreactors on a large scale, can meet production requirements of slow virus liquid, is stable and is good in repeatability; furthermore, the purified slow virus liquid prepared by virtue of the method is not polluted by exogenous factors, is low in residues of cell host protein and host DNA, good in safety, and high in purity and titer and can completely meet requirements of clinic gene therapy.

The invention discloses a bioreactor cell culture quality evaluation method, and relates to the technical field of bioreactors. The bioreactor cell culture quality evaluation method comprises image enhancement, image segmentation, smoothing processing, connected region extraction, standard cell area judgment and cell distribution statistics, and completes automatic evaluation of the cell culture quality through statistics of the number of cells. According to the bioreactor cell culture quality evaluation method, the growth state of the cultured cells can be automatically evaluated, and a reliable guarantee means is provided for promoting the application of a large-scale cell culture technology in a cell factory.

The invention belongs to the technical field of biopharming, and discloses a method for preparing a chicken Marek's disease turkey herpes virus Fc126 strain live vaccine by utilizing a cell factory and the turkey herpes virus vaccine (Fc126 strain) prepared by utilizing the method. The turkey herpes virus vaccine (Fc126 strain) prepared in the cell factory is good in quality, high in immunization potency and stable in immunization effect. In addition, the turkey herpes virus vaccine (Fc126 strain) has the advantages of being low in generation, high in content of plaque forming unit (PFU), low in cost, simple in operation technology, easy to control and the like.

The invention discloses preparation of a diploid cell attenuated live vaccine from a measles long-47 strain and a preparation process thereof. A measles attenuated live vaccine is prepared from a diploid cell strain, a virus culture vessel serves as a cell factory, and a virus solution can be collected continuously for three times and is stored in the form of concentration solution. The diploid cell attenuated live vaccine passes a monkey neurovirulence test and a safety test. The technology belongs to a great reform on measles attenuated live vaccine production, and plays a qualitative role in improving the production quality of the measles attenuated live vaccine. According to the invention, exogenous factor pollution and the possibility of allergic reaction on a few people existing in chick embryo cell (CEC) production are avoided, and the problems of low production efficiency and low quality are solved.